1. Field of the Invention
The present invention relates generally to fluidic processing and, more particularly, to methods and apparatuses concerning an integrated fluidic device capable of enriching and isolating a suspect cell subpopulation from a suspension of cells and quantitatively analyzing that subpopulation for marker proteins and mRNAs for the purpose of detection and diagnosis of conditions such as cancer.
2. Description of Related Art
The identification of increasing numbers of genes that influence disease states and the approach of the post-genomic era make evident the need for faster and automated technologies that will allow dissemination of the gains of molecular diagnosis. If sufficiently small, automatic and inexpensive devices can be developed for molecular screening, they would not only revolutionize the diagnosis and prognosis of cancer and other diseases but also would enable molecular methods to be disseminated completelyxe2x80x94even to the point of care.
Although some devices such as gene chips and chip embodiments of the polymerase chain reaction (PCR) are beginning to enter use, many of the methods developed so far are labor intensive and are not readily suited to automated, continuous monitoring, or high throughput applications. Clearly, a wide range of enabling technologies is needed before integrated instruments capable of automated sample preparation and molecular analysis of clinical samples become a reality.
Technology that is the subject of the present addresses issues related to the creation of multiple-use diagnostic systems for combined sample preparation and detection of molecular markers. Disclosed herein are systems, methods, and devices capable of performing fully automated assays. These devices offer the advantages of small size, low sample volume requirements, and the potential for mass production at low cost. Such low-cost systems are applicable to reusable or disposable medical devices.
In one embodiment, such a system may include the following subsystems: (1) a prefilter stage to concentrate suspect cells; (2) a high discrimination separator stage to fractionate cell subpopulations; (3) a stage to burst cells and mobilize molecular components; and (4) a stage for automated analysis of protein and mRNA molecular diagnostic markers.
Important technologies for the development of such a system, and others made apparent by the present disclosure include the following: a prefiltering methodology to trap suspected cancer cells from blood or dispersed lymph node cells; a force balance method that exploits dielectric properties of the suspect cells, and, if needed, their immunomagnetic labeling properties, to fractionate them into a microfluidic isolation and analysis chamber; and a dielectric indexing and manipulation method for carrier beads that, when combined with certain established molecular assay methods, allows for the parallel quantification of multiple molecular markers.
As certain technology disclosed herein builds upon work involving dielectrophoretic trapping, dielectrophoretic field-flow fractionation (DEP-FFF), traveling wave methods, and other work performed by the inventors, the following are hereby specifically incorporated by reference herein in their entirety: U.S. Pat. No. 5,993,630 entitled xe2x80x9cMethod and Apparatus for Fractionation Using Conventional Dielectrophoresis and Field Flow Fractionationxe2x80x9d; U.S. Pat. No. 5,858,192 entitled xe2x80x9cMethod and Apparatus for Manipulation Using Spiral Electrodesxe2x80x9d; U.S. Pat. No. 5,888,370 entitled xe2x80x9cMethod and Apparatus for Fractionation Using Generalized Dielectrophoresis and Field Flow Fractionationxe2x80x9d; U.S. Pat. No. 5,993,632 entitled xe2x80x9cMethod and Apparatus for Fractionation Using Generalized Dielectrophoresis and Field Flow Fractionationxe2x80x9d; U.S. application Ser. No. 09/249,955 filed Feb. 12, 1999 and entitled xe2x80x9cMethod and Apparatus for Programmable Fluidic Processingxe2x80x9d now U.S. Pat. No. 6,294,063; U.S. application Ser. No. 09/395,890 filed Sep. 14, 1999 and entitled xe2x80x9cMethod and Apparatus for Fractionation Using Generalized Dielectrophoresis and Field Flow Fractionationxe2x80x9d, now U.S. Pat. No. 6,287,832; U.S. Provisional Application No. 60/211,757 filed Jun. 14, 2000 and entitled xe2x80x9cMethod and Apparatus for Combined Magnetophoretic and Dielectrophoretic Manipulation of Analyte Mixturesxe2x80x9d; U.S. Provisional Application No. 60/211,515 filed Jun. 14, 2000 and entitled xe2x80x9cDielectrically-Engineered Microparticlesxe2x80x9d; U.S. Provisional Application No. 60/211,516 filed Jun. 14, 2000 and entitled xe2x80x9cApparatus and Method for Fluid Injection.xe2x80x9d
Dielectric indexing represents a new approach to identifying individual molecular tests in a parallel molecular analysis scheme that substitutes dielectric indexing of carrier beads for the spatial indexing used on a gene chip. This new approach allows different subpopulations of beads, each carrying a probe of a different molecular marker, to be identified and manipulated within the carrier medium using a dielectric fingerprint unique to each bead/probe type. The need to immobilize different molecular probes in a tightly specified pattern on a fixed substrate as demanded, for example, by gene chip technology, is thereby eliminated. Mixtures of probes, each probe carried on a separately indexed bead type, may be injected into and flushed from a reusable assay system in order to examine any desired panel of molecular markers.
The use of bead dielectric properties as an indexing parameter not only provides the capability of manipulating beads through dielectrophoresis or another suitable manipulation force, but also offers a new alternative to optical or fluorescent bead indexing methods that might interfere with low light emissions in fluorescent probe assays.
Technology disclosed herein builds upon and synthesizes aspects of many disciplines including field-flow fractionation (physical chemistry), dielectrophoresis and magnetophoresis (physics), microfluidics (mechanical and fluid engineering), microfabrication (photolithography, MEMS and magnetic materials science), control electronics (electrical engineering), antibody and nucleic acid binding and linking (immunology and molecular biology), cell biology (cell culture and cytology), flow cytometry, and oncology.